Biology subjects

Lan, Y.

Publications and source records attributed to Lan, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Host mixotrophy, habitat stability and recombination may all decrease geographic differentiation between symbiont populations in Western Pacific deep-sea mussels

The intra-host composition of horizontally transmitted microbial symbionts can vary across host populations due to interactive effects of host genetics, environmental and geographic factors. While adaptation to local habitat conditions can drive geographic subdivision of symbiont strains, it is unknown whether these patterns are universal and how differences in ecological characteristics among host-symbiont associations influence the genomic structure of symbiont populations. To address this question, we sequenced metagenomes of different populations of the deep-sea mussel Bathymodiolus septemdierum, which are common at Western Pacific deep-sea hydrothermal vents and show characteristic patterns of niche partitioning with sympatric gastropod symbioses. Bathymodiolus septemdierum lives in close symbiotic relationship with sulfur-oxidizing chemosynthetic bacteria but supplements its symbiotrophic diet through filter-feeding, enabling it to occupy ecological niches with less exposure to geochemical reductants. Our analyses indicate that symbiont populations associated with B. septemdierum show structuring by geographic location, but that the dominant symbiont strain is uncorrelated with vent site. These patterns are in contrast to co-occurring Alviniconcha and Ifremeria symbioses that exhibit greater symbiont nutritional dependence and occupy habitats with higher spatial variability in environmental conditions. Our results suggest that relative habitat homogeneity combined with sufficient symbiont dispersal and genomic mixing might promote persistence of similar symbiont strains across geographic locations, while mixotrophy might decrease selective pressures on the host to affiliate with locally adapted symbiont strains. Overall, these data contribute to our understanding of the potential mechanisms influencing symbiont population structure across a spectrum of marine microbial symbioses that vary in ecological niche and relative host dependency.

evolutionary biology↗

bioRxiv2022

HDL-C as a potential medium between depletion of Lachnospiraceae genera and hypertension under high-calorie diet

Gut microbial dysbiosis has been associated with hypertension. An extremely high incidence of essential hypertension was found in the Han and the Yugur who resided in Sunan county in East Asias nomadic steppes with little population movement. In attempt to investigate the gut microbial role in hypertension, we recruited a total of 1, 242 Yugur and Han people, who had resided in Sunan County for more than 15 years and accounted for 3% of the local population. The epidemiological survey of 1,089 individuals indicated their nearly 1.8 times higher prevalence of hypertension (38.2-43.3%) than the average in China (23.2%), under a special high-calorie diet based on wheat, cattle, mutton, and animal offal. The 16S rRNA gene sequencing on the fecal samples of 153 individuals revealed that certain Lachnospiraceae genera were negatively correlated with high-density lipoprotein cholesterol (HDL-C, P = 5.46 x 10-6), systolic blood pressure (SBP, P = 7.22 x 10-3), diastolic blood pressure (DBP, P = 1.8 x 10-3). HDL-C was positively correlated with SBP (P = 0.023). We further observed that serum butyrate content was lower in both Han (P = 1.99 x 10-3) and Yugur people (P = 0.031) with hypertension than those without hypertension. This study gives a novel insight into the role of gut microbial dysbiosis in hypertension modulation under a high-calorie diet, where the notable depletion of Lachnospiraceae genera might lead to less production of butyrate, contributing to the lower level of HDL-C, and elevating blood pressure in hypertension. IMPORTANCEDietary nutrients can be converted by gut microbiota into metabolites such as short-chain fatty acids, which may serve as disease-preventing agents in hypertension. Due to limited population mobility and a unique high-calorie dietary habit, the recruited cohort in this study could be a representative for elucidating the associations between gut microbiota and hypertension under high-calorie diet. Moreover, low levels of HDL-C have previously been associated with an increased risk of various cardiovascular diseases (CVDs). Our findings provide a new insight that low levels of HDL-C may be a potential medium between depletion of Lachnospiraceae genera and hypertension under high-calorie diet, which might also be a potential candidate for other CVDs.

microbiology↗

bioRxiv2022

Spurious intragenic transcription is a hallmark of mammalian cellular senescence and tissue aging

Mammalian aging is characterized by the progressive loss of tissue integrity and function manifesting in ill health and increased risk for developing multiple chronic conditions. Accumulation of senescent cells in aging tissues partly contributes to this decline and targeted depletion of senescent cells in vivo ameliorates many age-related phenotypes. However, the fundamental molecular mechanisms responsible for the decline of cellular health and fitness during senescence and aging are largely unknown. In this study, we investigated whether chromatin-mediated loss of transcriptional fidelity, known to contribute to fitness and survival in yeast and worms, also occurs during human cellular senescence and mouse aging. Our findings reveal that aberrant transcription initiation inside genes is widespread in senescence and aging. It co-occurs with changes in the chromatin landscape and formation of non-canonical transcription start sites. Interventions that alter spurious transcripts have dramatic consequences on cellular health primarily affecting intracellular signal transduction pathways. We propose that spurious transcription is a conserved hallmark of aging that promotes a noisy transcriptome and degradation of coherent transcriptional networks.

genomics↗

bioRxiv2022

NIPBL and WAPL balance cohesin activity to regulate chromatin folding and gene expression

The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. We investigated the roles of NIPBL and WAPL, two regulators of cohesin activity, in chromatin folding and transcription in human cells. Consistent with their opposing roles in cohesin regulation, depletion of these factors showed opposite effects on levels of chromatin-bound cohesin and spatial insulation of neighboring domains. We find that NIPBL or WAPL depletion each alter the expression of ~2,000 genes, most of which are uniquely sensitive to either regulator. We find that each set of differentially expressed genes are enriched at chromatin loop anchors and clustered within the genome, suggesting there are genomic regions sensitive to either more or less cohesin. Remarkably, co-depletion of both regulators rescued chromatin misfolding and gene misexpression compared to either single knockdown. Taken together, we present a model in which the relative, rather than absolute, levels of NIPBL and WAPL are required to balance cohesin activity in chromatin folding to regulate transcription.

genetics↗

bioRxiv2022

Multi-trait genomic prediction improves selection accuracy for enhancing seed mineral concentrations in pea (Pisum sativum L.)

The superiority of multi-trait genomic selection (MT-GS) over univariate genomic selection (UNI-GS) can be improved by redesigning the phenotyping strategy. In this study, we used about 300 advanced breeding lines from North Dakota State University (NDSU) pulse breeding program and about 200 USDA accessions evaluated for ten nutritional traits to assess the efficiency of sparse testing in MT-GS. Our results showed that sparse phenotyping using MT-GS consistently outperformed UNI-GS when compared to partially balanced phenotyping using MT-GS. This strategy can be further extended to multi-environment multi-trait GS to improve prediction performance and reduce the cost of phenotyping and time-consuming data collection process. Given that MT-GS relies on borrowing information from genetically correlated traits and relatives, consideration should be given to trait combinations in the training and prediction sets to improve model parameters estimate and ultimately prediction performance. Our results point to heritability and genetic correlation between traits as possible parameters to achieve this objective.

genetics↗

bioRxiv2022

High-throughput Oligopaint screen identifies druggable regulators of genome folding

Although the molecular rules governing genome organization are being quickly elucidated, relatively few proteins regulating this process have been identified. To address this gap, we developed a fully automated imaging pipeline, called HiDRO (high-throughput DNA or RNA labeling with optimized Oligopaints), that permits quantitative measurement of chromatin interactions across a large number of samples. Using HiDRO, we screened the human druggable genome and identified >300 factors that regulate chromatin folding during interphase, including 43 validated hits that either increase or decrease interactions between topological associating domains (TADs). We discovered that genetic or chemical inhibition of the ubiquitous kinase GSK3A enhances long-range interactions by dysregulating cohesin-mediated chromatin looping. Collectively, these results highlight a noncanonical role for GSK3A signaling in nuclear architecture and underscore the broader utility of HiDRO-based screening to identify novel mechanisms that drive the spatial organization of the genome.

genetics↗

bioRxiv2022

Dysregulated H19/Igf2 expression disrupts cardiac-placental axis during development of Silver Russell Syndrome-like mouse models

Dysregulation of the imprinted H19/IGF2 locus can lead to Silver-Russell Syndrome (SRS) in humans. However, the mechanism of how abnormal H19/IGF2 expression contributes to various SRS phenotypes remains unclear, largely due to incomplete understanding of the developmental functions of these two genes. We previously generated a mouse model with humanized H19/IGF2 ICR (hIC1) on the paternal allele that exhibited H19/Igf2 dysregulation together with SRS-like growth restriction and perinatal lethality. Here we dissect the role of H19 and Igf2 in cardiac and placental development utilizing multiple mouse models with varying levels of H19 and Igf2. We report severe cardiac defects such as ventricular septal defects (VSDs) and thinned myocardium, placental anomalies including thrombosis and vascular malformations, together with growth restriction in mouse embryos that correlated with the extent of H19/Igf2 dysregulation. Transcriptomic analysis using cardiac endothelial cells of these mouse models shows that H19/Igf2 dysregulation disrupts pathways related to extracellular matrix (ECM) and proliferation of endothelial cells. Our work links the heart and placenta through regulation by H19 and Igf2, demonstrating that accurate dosage of both H19 and Igf2 is critical for normal embryonic development, especially related to the cardiac-placental axis.

developmental biology↗

bioRxiv2022

Pericentriolar matrix integrity relies on cenexin and Polo-Like Kinase (PLK)1

Polo-Like-Kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosomes pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa) and zebrafish embryos in parallel to phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation and that a conserved C-terminal PLK1 binding site between humans and zebrafish is needed for PCM maintenance through mediating PLK1-dependent substrate phosphorylation events. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexins C-terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to anchor PLK1 moderating its potential to phosphorylate PCM substrates required for PCM maintenance and function.

cell biology↗

bioRxiv2021

SARS-CoV-2 ORF8 encoded protein contains a histone mimic, disrupts chromatin regulation, and enhances replication

SARS-CoV-2 emerged in China at the end of 2019 and caused the global pandemic of COVID-19, a disease with high morbidity and mortality. While our understanding of this new virus is rapidly increasing, gaps remain in our understanding of how SARS-CoV-2 can effectively suppress host cell antiviral responses. Recent work on other viruses has demonstrated a novel mechanism through which viral proteins can mimic critical regions of human histone proteins. Histone proteins are responsible for governing genome accessibility and their precise regulation is critical for a cells ability to control transcription and respond to viral threats. Here, we show that the protein encoded by ORF8 (Orf8) in SARS-CoV-2 functions as a histone mimic of the ARKS motif in histone 3. Orf8 is associated with chromatin, binds to numerous histone-associated proteins, and is itself acetylated within the histone mimic site. Orf8 expression in cells disrupts multiple critical histone post-translational modifications (PTMs) including H3K9ac, H3K9me3, and H3K27me3 and promotes chromatin compaction while Orf8 lacking the histone mimic motif does not. Further, SARS-CoV-2 infection in human cell lines and postmortem patient lung tissue cause these same disruptions to chromatin. However, deletion of the Orf8 gene from SARS-CoV-2 largely blocks its ability to disrupt host-cell chromatin indicating that Orf8 is responsible for these effects. Finally, deletion of the ORF8 gene affects the host-cell transcriptional response to SARS-CoV-2 infection in multiple cell types and decreases the replication of SARS-CoV-2 in human induced pluripotent stem cell-derived lung alveolar type 2 (iAT2) pulmonary cells. These findings demonstrate a novel function for the poorly understood ORF8-encoded protein and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Finally, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

genetics↗

bioRxiv2021

Cleavage of histone H2A during embryonic stem cell differentiation destabilizes nucleosomes to counteract gene activation

Histone proteolysis is a poorly understood phenomenon in which the N-terminal tails of histones are irreversibly cleaved by intracellular proteases. During development, histone post-translational modifications are known to orchestrate gene expression patterns that ultimately drive cell fate decisions. Therefore, deciphering the mechanisms of histone proteolysis is necessary to enhance the understanding of cellular differentiation. Here we show that H2A is cleaved by the lysosomal protease Cathepsin L during ESCs differentiation. Using quantitative mass spectrometry (MS), we identified L23 to be the primary cleavage site that gives rise to the clipped form of H2A (cH2A), which reaches a maximum level of ~1% of total H2A after four days of differentiation. Using ChIP-seq, we found that preventing proteolysis leads to an increase in acetylated H2A at promoter regions in differentiated ES cells. We also identified novel readers of different acetylated forms of H2A in pluripotent ES cells, such as members of the PBAF remodeling complex. Finally, we showed that H2A proteolysis abolishes this recognition. Altogether, our data suggests that proteolysis serves as an efficient mechanism to silence pluripotency genes and destabilize the nucleosome core particle.

developmental biology↗

bioRxiv2021

Synthesis and characterization of 5-(2-fluoro-4-pyridine-7-carboxamide as a PET imaging ligand for metabotropic glutamate receptor 2

Metabotropic glutamate receptor 2 (mGluR2) is a therapeutic target for the treatment of several neuropsychiatric disorders and conditions. The role of mGluR2 function in etiology could be unveiled by in vivo imaging using positron emission tomography (PET). In this regard, 5-(2- fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7- carboxamide ([11C]13), a potent negative allosteric modulator (NAM), was developed to support this endeavor. Radioligand [11C]13 was synthesized via the O-[11C]methylation of phenol 24 with a high molar activity of 212 {+/-} 76 GBq/{micro}mol (n = 5) and excellent radiochemical purity (> 99%). PET imaging of [11C]13 in rats demonstrated its superior brain heterogeneity, particularly in the regions of striatum, thalamus, hippocampus, and cortex. Accumulation of [11C]13 in these regions of interest (ROIs) was reduced with pretreatment of mGluR2 NAMs, VU6001966 (9) and MNI-137 (26), the extent of which revealed a time-dependent drug effect of the blocking agents. In a nonhuman primate, [11C]13 selectively accumulated in mGluR2-rich regions, especially in different cortical areas, putamen, thalamus, and hippocampus, and resulted in high-contrast brain images. The regional total volume of distribution (VT) estimates of [11C]13 decreased by 14% after the pretreatment with 9. Therefore, [11C]13 is a potential candidate for translational PET imaging studies of mGluR2 function.

neuroscience↗

bioRxiv2021

CTCF and transcription influence chromatin structure re-configuration after mitosis

During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. Here we exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to re-sculpting the genome in newborn nuclei. Depletion of CTCF specifically during the M- to G1-phase transition altered the re-establishment of local short-range compartmentalization after mitosis. Chromatin domain boundary reformation was impaired upon CTCF loss, but a subset ([~]27%) of boundaries, characterized by transitions in chromatin states, was established normally. Without CTCF, structural loops failed to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persisted deeply into G1-phase in CTCF depleted cells. CTCF loss-associated gains in transcription were often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declined upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitated formation CRE loops nested within them, especially those involving weak CREs. Transcription inhibition did not elicit global architectural changes and left transcription start site-associated boundaries intact. However, ongoing transcription contributed considerably to the formation of gene domains, regions of enriched contacts spanning the length of gene bodies. Notably, gene domains formed rapidly in ana/telophase prior to the completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yielded novel insights into how CTCF and transcription contribute to the dynamic re-configuration of chromatin architecture during the mitosis to G1 phase progression.

genomics↗

bioRxiv2021

OsciDrop: A versatile on-demand droplet generator

Droplet microfluidics is a powerful tool in many biological and clinical applications. Microfluidic chips, such as flow-focusing droplet generators, have been extensively used to high-throughput encapsulate reactions with single-cell and single-molecular resolutions. However, microfabrication is expensive and precision-demanding, preventing it from widespread use in biomedical laboratories and clinical facilities. Herein, we present a versatile chip-free droplet generator, OsciDrop, for generating size-tunable droplets on demand, with high uniformity. OsciDrop segments the fluid flowing out of the orifice of a micropipette tip into droplets by oscillating the tip under the surface of a continuous oil phase. We investigated the factors influencing droplet generation by examining several control parameters. Results show that flow rate, oscillating amplitude, and frequency are key parameters to generate monodisperse droplets on demand. And OsciDrop is able to generate droplets in a flexible and repeatable manner. Importantly, using an optimal asymmetrical oscillation waveform, OsciDrop can controllably generate monodisperse droplets spanning a wide volume range (200 pL - 2 L). To demonstrate the ability of OsciDrop for chip-free droplet assays, a digital loop-mediated isothermal amplification (dLAMP) was performed to absolutely quantify African swine fever virus (ASFV). The OsciDrop method opens up a feasible and versatile avenue to perform droplet-based assays, exhibiting full accessibility for chip-free droplet microfluidics.

bioengineering↗

bioRxiv2021

Microfluidic Streptomyces Cultivation for Whole Lifecycle Characterization and Phenotypic Assays Enabled by Nanogap-stabilized Air-Water Interface

Streptomyces is a model filamentous prokaryote to study multicellular differentiation and a rich reservoir for antibiotics discovery. In their natural conditions, Streptomyces grows at the interface of porous soil, air, and water. The morphological development of Streptomyces is traditionally performed on agar plates and mostly studied at the population levels. However, the detailed lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools which can mimic their natural conditions in the soil. Here, we developed a simple assembled microfluidic device for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level. The microfluidic device composed of a microchannel for loading samples and supplying nutrients, microwell arrays for seeding and growth of single spores, and air-filled chambers aside of the microwells that facilitate growth of aerial hyphae and spores. A unique feature of this device is that each microwell is surrounded by a 1.5 {micro}m gap connected to an air-filled chamber which provide stabilized water-air interface. We used this device to observe the development of single Streptomyces spores and found that unlike those in bulk liquid culture, Streptomyces can differentiate at water-air interfaces in microscale liquid culture. Finally, we demonstrated that phenotypic A-Factor assay can be performed at defined time point of its lifecycle. This microfluidic device could become a robust tool for studying Streptomyces multi-cellular differentiation and interaction at single cell level. IMPORTANCEWe describe a microfluidic device that mimics the natural porous environment for the growth and development of Streptomyces, the model system for bacterial multicellularity. The microfluidic device is used for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level, including growth of aerial filaments. The aerial hyphae development of Streptomyces at the water-air interface was observed at real time in the microfluidic device. The early growth, opportunistic transformation (in the gap), and merging of aerial hyphae of Streptomyces in the microfluidic device were observed for the first time. It will play an important role in finding single-cell heterogeneity to study secondary metabolites related to the complex lifecycle of Streptomyces.

developmental biology↗

bioRxiv2021

Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples

BackgroundAdaptive sampling is a method of software-controlled enrichment unique to nanopore sequencing platforms recently implemented in Oxford Nanopores own control software. By examining the first few hundred bases of a DNA molecule as it passes through a pore, software can determine if the molecule is sufficiently interesting to sequence in its entirety. If not, the molecule is ejected from the pore by reversing the voltage across it, freeing the pore for a new molecule. User supplied sequences define the targets to be sequenced or ejected. Here we explore the potential of using adaptive sampling for enrichment of rarer species within metagenomic samples. ResultsWe created a synthetic mock community consisting of seven bacterial species at different proportions ranging from 1.2% to 47% and used this as the basis for a series of enrichment and depletion experiments. To investigate the effect of DNA length on adaptive sampling efficiency, we created sequencing libraries with mean read lengths of 1.7 kbp, 4.7 kbp, 10.6 kbp, and 12.8 kbp and enriched or depleted for individual and multiple species over a series of sequencing runs. Across all experiments enrichment ranged from 1.67-fold for the most abundant species with the shortest read length to 13.87-fold for the least abundant species with the longest read length. Factoring in the reduction to sequence output associated with repeatedly rejecting molecules reduces the calculated efficiency of this enrichment to between 0.96-fold and 4.93-fold. We note that reducing ejections due to false negatives (approximately 36%) would significantly increase efficiency. We used the relationship between abundance, molecule length and enrichment factor to produce a mathematical model of enrichment based on molecule length and relative abundance, whose predictions correlated strongly with experimental data. A web application is provided to allow researchers to explore model predictions in advance of performing their own experiments. ConclusionsOur data clearly demonstrates the benefit for enriching low abundant species in adaptive sampling metagenomic experiments, especially with longer molecules, and our mathematical model can be used to determine whether a given experimental DNA sample is suitable for adaptive sampling. Notably, repeated voltage reversals have no effect on pore stability.

genomics↗

bioRxiv2021

Fascin limits Myosin activity within Drosophila border cells to control substrate stiffness and promote migration

A key regulator of collective cell migrations, which drive development and cancer metastasis, is substrate stiffness. Increased substrate stiffness promotes migration and is controlled by Myosin. Using Drosophila border cell migration as a model of collective cell migration, we identify, for the first time, that the actin bundling protein Fascin limits Myosin activity in vivo. Loss of Fascin results in: increased activated Myosin on the border cells and their substrate, the nurse cells; decreased border cell Myosin dynamics; and increased nurse cell stiffness as measured by atomic force microscopy. Reducing Myosin restores on-time border cell migration in fascin mutant follicles. Further, Fascins actin bundling activity is required to limit Myosin activation. Surprisingly, we find that Fascin regulates Myosin activity in the border cells to control nurse cell stiffness to promote migration. Thus, these data shift the paradigm from a substrate stiffness-centric model of regulating migration, to uncover that collectively migrating cells play a critical role in controlling the mechanical properties of their substrate in order to promote their own migration. This new means of mechanical regulation of migration is likely conserved across contexts and organisms, as Fascin and Myosin are common regulators of cell migration.

cell biology↗

bioRxiv2020

Dissecting human skeletal stem cell ontogeny by single-cell transcriptomic and functional analyses

Human skeletal stem cells (SSCs) have been discovered in fetal and adult bones. However, the spatiotemporal ontogeny of human SSCs during embryogenesis has been elusive. Here we map the transcriptional landscape of human embryonic skeletogenesis at single-cell resolution to address this fundamental question. We found remarkable heterogeneity within human limb bud mesenchyme and epithelium, as well as the earliest osteo-chondrogenic progenitors. Importantly, embryonic SSCs (eSSCs) were found in the perichondrium of human long bones, which self-renew and generate osteochondral lineage cells, but not adipocytes or hematopoietic stroma. eSSCs are marked by the adhesion molecule CADM1 and highly enrich FOXP1/2 transcriptional network. Interestingly, neural crest-derived cells with similar phenotypic markers and transcriptional network were also found in the sagittal suture of human embryonic calvaria. Taken together, this study revealed the cellular heterogeneity and lineage hierarchy during human embryonic skeletogenesis, and identified distinct skeletal stem/progenitor cells that orchestrate endochondral and intramembranous ossification.

developmental biology↗

bioRxiv2020

Uncovering the Structure and Function of Microbial Communities Formed During Periodic Tilling of TNT and DNT Co-Contaminated Soils

1.Environmental contamination by 2,4,6-trinitrotoluene (TNT), historically the most widely used secondary explosive, is a long-standing problem in former military conflict areas and at manufacturing and decommissioning plants. In field test plots at a former explosives manufacturing site, removal of TNT and dinitrotoluenes (DNTs) was observed following periods of tillage. Since tilling of soils has previously been shown to alter the microbial community, this study was aimed at understanding how the microbial community is altered in soils with historical contamination of nitro explosives from the former Barksdale TNT plant. Samples of untilled pristine soils, untilled TNT-contaminated soils and tilled TNT-contaminated soils were subjected to targeted amplicon sequencing of 16S ribosomal RNA genes in order to compare the structure of their bacterial communities. In addition, metagenomic data generated from the TNT tilled soil was used to understand the potential functions of the bacterial community relevant to nitroaromatic degradation. While the biodiversity dropped and the Burkholderiales order became dominant in both tilled and untilled soil regardless of tillage, the bacterial community composition at finer taxonomic levels revealed a greater difference between the two treatments. Functional analysis of metagenome assembled genome (MAG) bins through systematic review of commonly proposed DNT and TNT biotransformation pathways suggested that both aerobic and anaerobic degradation pathways were present. A proposed pathway that considers both aerobic and anaerobic steps in the degradation of TNT in the scenario of the tilled contaminated soils is presented. 2. ImportanceIn this study, TNT and DNT removal has been observed in field-scale experiments following periodic tilling of historically contaminated soils. The microbial community structures of uncontaminated pristine soils, untilled contaminated soils, and tilled contaminated soils were investigated using high-throughput sequencing platforms. In addition, shotgun metagenome libraries of samples from tilled contaminated soils were generated. The results indicated that a significant shift of the bacterial community at the family level between tilled and untilled contaminated soils, with tilled soils being dominated by Alcaligenaceae and untilled soils by Burkholderiacea. In-depth metagenomic analysis of samples from tilled contaminated soils, indicate the presence of genes that encode for enzymes that potentially could lead to mineralization of TNT and DNT under mixed aerobic and anaerobic periods.

genomics↗